Lumazine synthase catalyzes, the condensation of 5-amino-6-(D-ribitylamino)-2,4-(1H,3H)pyrimidinedione with the novel carbohydrate, L-3,4-dihydroxy-2-butanone-4-phosphate to form the immediate precursor to riboflavin, 6,7-dimethyl-8-(D-ribitylamino)lumazine. Recent developments in our understanding of lumazine synthase include the identification of L-3,4-dihydroxy-2-butanone-4-phosphate as one of the substrates, and the X-ray structure of reconstituted beta 60 capsids of lumazine synthase complexed with 5-(6-D-ribitylamino-2,4-dihydroxypyrimidin-5-yl)-1-pentylphosphonic acid, a metabolically stable analog of the hypothetical Schiff base intermediate in the enzyme-catalyzed reaction. The identification of the carbohydrate precursor has allowed a mechanistic hypothesis to be advanced concerning the pathway for the conversion of the substrates into the lumazine product. In addition, the X-ray structure has made it possible to propose a mechanism for how the enzyme functions, as well as a hypothetical binding geometry for the substrates and proposed reaction intermediates. In order to test these new ideas, metabolically stable analogs of the hypothetical reaction intermediates are required. The present research project involves the design and synthesis of a set of metabolically stable reaction intermediate analogs that will be valuable in obtaining evidence about the structural and mechanistic details of lumazine synthase catalysis, including protein and ligand mobility during the catalytic event. These ligands will be bound to the active site of lumazine synthase by ligand driven aggregation or by diffusion and the X-ray structures of the resulting complexes will be determined. The distances of certain atoms of the ligands to 15N nuclei of the protein will be determined to within 0.2 A by REDOR NMR, and these distances will be used in combination with the X-ray crystal coordinates in a series of distance-restrained molecular dynamics simulations to obtain a binding model for each ligand. All potential mutations of the protein can be made by site-directed mutagenesis, and the recombinant protein expressed in E. coli. The mechanistic interpretation will be based on protein as well as ligand modifications. Uniformly 15N-labeled protein and fluorine-containing protein will be especially useful in the interpretation of the REDOR spectra. The recent identification of L-3,4-dihydroxy-2-butanone-4-phosphate as a substrate of lumazine synthase has made it possible to determine the standard thermodynamic and kinetic parameters of lumazine synthase inhibitors. The inhibition constants (Ki) and dissociation constants (Kd) of the new inhibitors will be determined, and ligand displacement studies will also be performed. The binding stochiometry will be determined. These experiments will be performed on both hollow beta 60 capsids and heavy riboflavin synthase.